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The SRC gene was the first proto-oncogene to be discovered. Its product SRC is a nonreceptor protein tyrosine kinase that is the prototype, and a ubiquitously expressed member, of the SRC family kinases. SRC has been investigated for decades in mouse and in vitro models: these studies have indicated that SRC signalling has a central role in many cellular functions and in oncogenesis. In platelets, SRC mediates signal activation pathways downstream different integrins and G protein-coupled receptors. However, much remains to be understood about SRC functions in human megakaryocytes and platelets. Recently, the first germline mutation in SRC causing human disease was reported in two families. The heterozygous c.1579G>A variant, causing the p.E527K substitution in SRC, was characterized as a gain-of-function mutation resulting in a constitutively active SRC due to the enhanced autophosphorylation of its Tyrosine-419. This mutation was associated with a complex syndromic phenotype characterized by thrombocytopenia variably associated with facial dysmorphism, severe osteoporosis, autism, intellectual disability, premature edentulism, and adult-onset myelofibrosis with splenomegaly. It has been suggested that thrombocytopenia derives from defective megakaryocyte maturation.Here we report the investigation of a new unrelated individual carrying the p.E527K variant that provides additional information on the clinical and pathogenetic features of the disorder and the role of SRC in human megakaryocytes. The study was approved by the Ethic Committee of Pavia and conducted according to the Declaration of Helsinki. The patient’s parents provided written informed consent. Clinical and laboratory characterization. The proband was a 2-year-old male, born to healthy parents, referred for the investigation of congenital thrombocytopenia and excessive bleeding. Platelet count ranged from 55100x10/L with no other cytopenias (Online Supplementary Table S1). He presented at age 3 days with bilateral cephalohematoma with jaundice, and since then has had a lifelong history of easy bruising, petechiae, and occasional moderate epistaxis. Moreover, at the age of 3 months he was diagnosed with spontaneous left periventricular hemorrhage resulting in mild right hemiparesis. Physical examination showed no pathological findings, except for the hemiparesis. Intellectual development and learning were normal. Examination of peripheral blood smears revealed platelet anisocytosis and macrocytosis and a reduced a-granules content with about 30% of hypoor agranular platelets (Online Supplementary Figure S1A-C). The a-granule defect was confirmed at ultrastructural analysis (Online Supplementary Figure S1D). Consistent with the a-granule deficiency, the platelet amount of thrombospondin-1 and von Willebrand factor was reduced (Online Supplementary Figure S1E-F), and the surface exposure of P-selectin was defective after stimulation with all tested agonists (Online Supplementary Figure S1G). Study of platelet aggregation showed a defective response to collagen, whereas response to ADP and TRAP was normal (Online Supplementary Table S2). Platelet functional response to these agonists was also investigated as the induction of surface expression of the activated form of glycoprotein IIbIIIa: we found a significantly reduced response to collagen and a very mild defect after stimulation with ADP and TRAP (Online Supplementary Figure S1H). Flow cytometry did not show any defects in the expression of the major platelet surface glycoproteins (Online Supplementary Table S3). In order to investigate the genetic cause of the thrombocytopenia, we performed whole exome sequencing of the proband. The analysis identified the c.1579G>A variant in the SRC gene (p.E527K substitution in SRC) in heterozygosis. No other potentially pathogenetic variants were found in the other genes known to be responsible for inherited thrombocytopenia. Sanger sequencing showed that the proband’s parents, who had normal blood cell counts, did not carry the variant, which was therefore considered as de novo. The clinical picture remained stable until the age of the 3.5 years, when the proband began to present unexplained, moderate hyporegenerative anemia (Online Supplementary Table S1). A bone marrow (BM) biopsy was therefore performed. Examination concluded for a hypercellular BM with moderate trilineage dysplasia, no excess blasts, and significantly increased reticulin (fibrosis grade 2 according to Thiele). Megakaryocytes (Mk) were numerous, irregularly distributed in intertrabecular areas, and represented at all stages of maturation with dysmegakaryopoiesis and some micromegakaryocytes. No signs of Mk emperipolesis were present. Subsequently, anemia progressively worsened and required periodic red blood cell transfusions; we observed also a progressive decrease in platelet count and a mild splenomegaly (Online Supplementary Table S1). The search for a donor for hematopoietic stem cell transplantation was therefore started. Pathogenetic studies. We first checked that the SRC p.E527K variant actually results in constitutive increased activation of SRC. Consistent with previous findings, SRC was in a hyperactivated state in the patient’s resting platelets (Online Supplementary Figure S2). We also found increased activation of the tyrosine kinase FAK, a substrate of SRC involved in several mechanisms, such as cell adhesion and cytoskeleton reorganization (Online Supplementary Figure S2). The experiments reported below were carried out on two different occasions, when the patient was 2.5 and 3.0 years old, and the results were compared with those obtained in three healthy controls. Given the suggested role of SRC in cell adhesion, we tested the ability of the patient platelets to interact with different proteins of the extracellular matrix (ECM). Mutant platelets showed significantly increased adhesion and spreading on fibrinogen, type I collagen, and von Willebrand Factor (Online Supplementary Figure S3), suggesting that SRC hyperactivation induces a generalized enhancement of platelet adhesion to the ECM. We then differentiated in vitro Mk from peripheral blood progenitors of the patient according to a standard protocol. At the end of the culture, the maturation profile of the patient’s Mk was similar to that of controls (Figure 1A-C). Differently from previous findings, proplatelet formation (PPF) of mutant Mk in suspension liquid cultures was comparable to controls (Figure 1D-E). However, when Mk were let adhere to fibrinogen or type I collagen, two components of the BM ECM that regulate platelet formation, mutant Mk exhibited a markedly increased adhesion and spreading, often with aberrant morphology (Figure 2; Online Supplementary Figure S4). This prominent adhesion phenotype was associated with

Pathogenetic and clinical study of a patient with thrombocytopenia due to the p.E527K gain-of-function variant of SRC